Patent Application
20230087263
The present invention relates to the field cancer immunotherapies based on bispecific antibodies or on T cells modified by genetic engineering for secreting bispecific antibodies. In particular, the present invention relates to bispecific antibodies comprising an anti-CD19 and an anti-CD3 domain and T cells secreting the same, useful for the treatment of hematological malignancies, such as lymphoma, leukemia and myeloma.
Several cancer immunotherapies based on the recognition of tumor-associated antigens (TAA) by T cells have emerged in the past few decades, such as therapies based on chimeric antigen receptors (CAR) or bispecific antibodies.
CAR is a synthetic receptor consisting of an extracellular antibody domain that recognizes a TAA and an intracellular signaling domain derived from the T cell receptor (TCR), most commonly CD3ζ (the zeta chain associated with the T cell receptor complex). Second- and third-generation CARs also include one or two co-stimulatory domains, respectively, usually derived from CD28 and/or 4-1BB. In CAR-T cell therapies, autologous T cells from the patient are manipulated ex vivo to express a CAR recognizing a TAA and are infused back into the patient.
Bispecific antibodies are engineered antibodies designed to recognize two different epitopes of the same or different antigens. Tandem single-chain fragment variable (scFv), also known as bispecific T cell engager (BiTE), consists of two scFv connected by a flexible linker on a single polypeptidic chain. Blinatumomab is an anti-CD19/anti-CD3 tandem scFv approved by the U.S. Food and Drug Administration (FDA) under the accelerated approval program for the treatment of relapsed/refractory B cell acute lymphoblastic leukemia (ALL) and minimal residual disease-positive B cell ALL.
Secretion of bispecific antibodies by T cells modified by genetic engineering combines aspects of antibody-based therapies and cell-based therapies. Bispecific antibodies secreted in the tumor by T cells modified by genetic engineering redirect non-engineered infiltrating T cells (bystander T cells) to tumor cells. In addition, cells engineered to produce bispecific antibodies located outside tumors have demonstrated potent systemic antitumor activity in mouse tumor models (Blanco et al, 2019).
T cells transduced by retrovirus and secreting an anti-CD3/anti-CD19 bispecific BiTE antibody in vitro recognized tumor cells and redirected infiltrating T cells (bystander T cells) to tumor cells and infusion of said T cells resulted in regression of leukemia and lymphoma in xenograft animal models (Velasquez et al, 2016).
T cells transduced by RNA electroporation and secreting an anti-CD3/anti-CD19 bispecific BiTE antibody has been disclosed by Liu et al. Said T cells were tested both in vitro and in a leukemia mouse model and compared with CD19 CAR-T cells. The T cells recognize CD19+ cells lines and has increased tumor killing ability, prolonged functional persistence, increased cytokine production and potent proliferation over CD19 CAR-T cells (Liu et al, 2016).
Anti-CD3 OKT3 single-chain fragment variable (scFv) (Iwahori et al, 2015) and a CAR comprising the anti-CD19 A3B1 scFv (Castella et al, 2019) have been disclosed in the prior art.
There is currently a need to develop improved cancer immunotherapies based on anti-CD19/anti-CD3 bispecific antibodies and on modified T cells by genetic engineering for secreting them. Said improved cancer immunotherapies provide substantial benefits to the treatment of cancer patients.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
Unless defined otherwise, all the technical and scientific terms have the same meaning as those commonly understood by a person skilled in the art in the field of the invention.
Throughout the description and the claims, the terms “comprises”, “comprising” and their variants are not limiting in nature and therefore do not aim to exclude other technical features.
Throughout the description and the claims, the terms “comprise”, “comprising” and their variants includes, specifically, the term “consisting” or “consisting of”. When referred to compounds or biological sequences, it can be understood that the compounds of the invention may “comprise” any of the sequences along with other sequences or molecules or, in a preferred embodiment, the compounds of the invention may “consist of” any of said sequences, the latter case meaning that the compounds of the invention are precisely restricted to the fragment identified as such by the sequence.
Amino acids are referred herein by their commonly known three letter symbols. The following three letter symbols are used herein: Ile, Ala, Ser, Asp, Thr and Gly. “Ile” refers to isoleucine, “Ala” refers to alanine, “Ser” refers to serine, “Asp” refers to aspartic acid, “Thr” refers to threonine and “Gly” refers to glycine.
An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminal domain of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain domains respectively.
The term “bispecific antibody” refers to an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as different target binding but includes as well binding to different epitopes in one target.
The term “single-chain fragment variable” (scFv) molecule refers to a molecule having a variable domain of light chain (VL) and a variable domain of heavy chain (VH) connected by a polypeptide chain, arranged in the order VL-VH or VH-VL.
The term “BiTE”, also known as “tandem single chain fragment variable (ta-scFv)”, refers to bispecific T cell engager and consists of two scFv connected by a flexible linker on a single polypeptidic chain.
An “anti-CD19/anti-CD3 bispecific antibody” refers to a bispecific antibody comprising two different antigen-binding regions, one of which binds specifically to the antigen CD19 and one of which binds specifically to CD3.
“CDR” refers to complementarity determining regions. Complementarity determining regions are part of the variable chains in antibodies, where these molecules bind to their specific antigen. There are three CDRs (CDR1, CDR2 and CDR3) on the amino acid sequence of a variable domain of an antibody.
By a protein (or antibody) or polynucleotide having a sequence at least, for example, 95% “identical” to a reference sequence of the present invention, it is intended that the sequence is identical to the reference sequence except that the sequence may include up to five different residues per each 100 residues of the reference sequence. In other words, to obtain a protein (or antibody) having an amino acid sequence at least 95% identical to a reference protein sequence, up to 5% of the amino acids in the reference sequence may be deleted or substituted with another amino acids, or a number of amino acids up to 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any protein (or antibody) or polynucleotide sequence is at least, for example, 95% identical to a sequence of the present invention can be determined conventionally using known computer programs.
The term “genetic engineering” refers to modification and editing the genome of mammalian cells by gene transfer and gene-editing technologies. Genetic engineering may be applied to T cells, as well as other immune cells, such as natural killer cells.
The term “transducing” refers to a process by which an exogenous polynucleotide is introduced into a host cell. A “transduced” cell is one which has been transduced with an exogenous polynucleotide. The cell includes the primary subject cell and its progeny.
“Chimeric Antigen Receptor” (CAR) refers to a synthetic receptor consisting of an extracellular antibody domain that recognizes a tumor-associated antigen (TAA) and an intracellular signaling domain derived from the T cell receptor (TCR), most commonly CD3 (the zeta chain associated with the T cell receptor complex). CAR may also include one or two co-stimulatory domains, usually derived from CD28 and/or 4-1BB.
“CAR-T cell treatment” refers to a treatment in which autologous T cells from a patient are modified ex vivo by genetic engineering to express a CAR recognizing a tumor-associated antigen (TAA) and are infused back into the patient.
The term “bystander T cell” refers to a T cell present at the tumor site that can be activated through a mechanism which is independent from its endogenous T cell receptor (TCR) specificity.
The term “STAb-T cell treatment” refers to a cancer therapy based on endogenous secretion of bispecific antibodies secreted by T cells modified by genetic engineering, in which said bispecific antibodies secreted in the tumor by T cells modified by genetic engineering redirect non-engineered infiltrating T cells (bystander T cells) to tumor cells. “STAb-T cells” refers to T cells modified by cell engineering for secreting bispecific antibodies.
The term “effective amount” refers to the amount of a molecule or cell, such as an antibody or cell, that provides an objectively identifiable improvement in the subject's condition, recognized by a qualified observer, wherein said patient treated with a pharmaceutical composition comprising said amount of the molecule or cell.
The term “vector” refers to a polynucleotide molecule that is used to introduce and direct the expression of a specific gene in a target cell. The term includes the vector as a self-replicating polynucleotide as well as the vector incorporated into the genome of a host cell into which it has been introduced. Once the vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery.
The term “transwell assay” refers to a test carried out in a plate comprising an upper well (insert well) and a bottom well, separated by a transwell membrane. There are several transwell membranes with pore sizes that range from 3 μm, 5 μm, and 8 μm, which are commercially available from companies such as Corning. If the cells are too large compared to the pore size of the transwell membrane, they don't pass through the pores.
The term “IL” refers to interleukin. Throughout the description and the claims, the following terms are used: IL-1, IL-2, IL-7 and IL-15. “IL-1” refers to the protein interleukin 1, “IL-2” refers to the protein interleukin 2, “IL-7” refers to the protein interleukin 7 and “IL-15” refers to the protein interleukin 15, and the like.
The technical problem to be solved is to provide improved anti-CD19/anti-CD3 bispecific antibodies and improved T cells modified by genetic engineering for secreting them which provide improved cancer immunotherapies.
The invention as defined in the claims provides a solution to this technical problem.
The present invention provides a bispecific antibody comprising:
In embodiments, the bispecific antibody of the invention also includes antigen binding fragments thereof, which refers to fragments of the bispecific antibody capable of binding to the antigens of the bispecific antibody of the invention, i.e. CD19 and CD3.
The bispecific antibody of the invention is based on the combination of the anti-CD19 A3B1 scFv and the anti-CD3 OKT3 scFv. The bispecific antibody of the invention unexpectedly provides an improved cytotoxic activity with respect to the bispecific antibodies disclosed in the prior art.
It is known that the OKT3 clone binds to a conformational epitope dominated by a linear stretch of residues present in one of the CD3ε subunit loops (Kjer-Nielsen et al, 2004) and both the bispecific antibody of the invention and blinatumomab recognized, with different affinities, a similar epitope located on the CD3ε subunit of the complex.
However, the bispecific antibody of the invention and blinatumomab recognize different epitopes. The HD37 clone of blinatumomab recognizes the same region as the anti-CD19 B43 clone, or an adjacent one. The crystal structure of the CD19-B43 complex revealed that the monoclonal antibody B43 binds an epitope located on a region distant to the cell membrane. The bispecific antibody of the invention binds to a region closer to the cell membrane.
The exact location of the epitope impacts the access of the bispecific antibodies to their binding region and relates to their cytotoxic activity. Epitopes located in regions distant to the cell membrane have easier access than epitopes located close to the cell membrane, since the latter would have to face several steric effects associated to the epitope's proximity to the cell membrane. Thus, HD37-based interaction, as it is far from the cell membrane, results in a more efficient binding; in contrast, binding of the bispecific antibodies of the invention, results in a less efficient binding, since the epitope is located in a region closer to the cell membrane.
It has been reported that bispecific antibodies recognizing epitopes located in regions distant to the cell membrane showed reduced cytotoxic activity (Bluemel et al, 2010).
The bispecific antibodies of the invention benefit from their membrane-proximal epitope hence resulting in increased cytotoxic activity.
The examples of the invention evidence that T cells according to the invention efficiently secrete a bispecific antibody according to the invention and said T cells successfully activate unstimulated T cells when co-cultured in the presence of CD19+, but not CD19-target cells.
In an embodiment of the bispecific antibody of the invention:
In a preferred embodiment of the bispecific antibody of the invention:
In a preferred embodiment, the bispecific antibody of the invention further comprises a signal peptide.
In a preferred embodiment of the bispecific antibody of the invention, said signal peptide is selected from the group consisting of human kappa light chain signal peptide, human lambda light chain signal peptide, human heavy chain signal peptide (VH1, VH2, VH3, VH4, VHS, VH6 or VH7), oncostatin M signal peptide, human interleukin signal peptides, such as IL-1 signal peptide and IL-2 signal peptide.
In a preferred embodiment, the bispecific antibody of the invention further comprises a linker.
Suitable nonimmunogenic linker peptides include (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser-Gly4)n or Gly2(Ser-Gly2)n linker peptides, wherein n is generally a number between 1 and 10, typically between 1 and 4.
In a preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence selected from the group consisting of (Gly4-Ser)n, (Ser-Gly4)n, (Gly4-S)n, Gly4(Ser-Gly4)n, Gly2(Ser-Gly2)n, wherein n is a value from 1 to 10. In a more preferred embodiment, said linker is a peptide linker consisting of the amino acid sequence (Gly4-Ser)n, wherein n is a value from 1 to 10. In a yet more preferred embodiment, n is a value from 1 to 4.
In other preferred embodiment, the bispecific antibody of the invention further comprises a polyhistidine tag.
The bispecific antibody comprising a polyhistidine tag enables carrying out identification of the bispecific antibody by agents recognizing the polyhistidine tag, such as specific antibodies for that tag. It also enables carrying out purification methods based on recognition of the polyhistidine tag, such as chromatographic methods, High Performance Liquid Chromatography (HPLC), etc.
In other preferred embodiment of the bispecific antibody of the invention, said bispecific antibody is at least 95% identical to the amino acid sequence SEQ ID NO: 1.
In a more preferred embodiment of the bispecific antibody of the invention, said bispecific antibody consists of the amino acid sequence SEQ ID NO: 1.
The present invention also provides an ex vivo method of preparation of T cells secreting the bispecific antibody of the invention, the method comprising transducing isolated T cells from a subject with a synthetic polynucleotide encoding the bispecific antibody of the invention, or a vector comprising the same.
There are various methods of introducing exogenous polynucleotides into a host cell. Physical treatments, chemical compounds, biological particles or combinations thereof can be used in said methods of introducing exogenous polynucleotides into a host cell.
Methods of introducing exogenous polynucleotides into a host cell include, but are not limited to, electroporation, microinjection, gene gun, cell squeezing, impalefection, sonoporation, optical transfection, hydrodynamic delivery, lipofection, magnetofection, particle bombardment, nucleoside-modified mRNA and RNA transfection.
Methods of introducing exogenous polynucleotides into a host cell include the use of DNA or RNA vectors. Vector for introducing exogenous polynucleotides into a host cell include, but are not limited to, transposon-based vectors, Sleeping Beauty transposon-based vectors and minicircles.
Chemical compounds for introducing exogenous polynucleotides into a host cell include, but are not limited to, calcium phosphate, highly branched organic compounds, such as dendrimers, cationic polymers, such as DEAE-dextran or polyethylenimine (PEI), liposomes and polymeric gene carriers (polyplexes).
Biological particles for introducing exogenous polynucleotides into a host cell include viral particles, which can be derived from lentivirus, retrovirus, poxvirus, adenovirus, adeno-associated virus and herpes simplex virus I. In an embodiment, viral vectors are lentiviral vectors.
In an embodiment of the method of preparation of T cells of the invention, the method comprises:
An automated cell processing platform may be used for processing T cells in all steps in which T cells of the invention are processed. Processing T cells includes culturing, activating, genetic modifying, selecting and expanding T cells. An automated cell processing platform enables the magnetic separation of different cells types, automated washing, fractionation and cultivation of cells. In an automated cell processing platform, cell processing steps may take place in a single-use chamber and may include specific tubing sets. Suitable automated cell processing platform include the CliniMACS Prodigy® platform, commercially available from Miltenyi Biotec.
In an embodiment of the method of preparation of T cells secreting the bispecific antibody of the invention, the method comprises:
In a preferred embodiment of the method of preparation of T cells secreting the bispecific antibody of the invention, the method comprises:
Biosafety of a vector production system is best achieved by distributing the sequences encoding its various components over as many independent units as possible, to maximize the number of recombination events that would be required to recreate a replication-competent virus. This ensures that only replication-defective viruses are produced, because the various plasmids would have to undergo multiple and complex recombination events to regenerate a replication-competent virus.
In the previous embodiment, lentiviral particles are produced using a 3-plasmid system, alternatively known as a second generation lentiviral vector.
The human immunodeficiency virus 1 (HIV-1) Rev protein is an accessory protein playing an important role on viral genome nuclear exportation.
The vesicular stomatitis virus (VSV) G envelope protein is a glycoprotein that interacts with target cell receptors to mediate virus cell entry.
Suitable lentiviral vectors encoding the bispecific antibody of the invention include pCSCMV. Suitable plasmids encoding the human immunodeficiency virus 1 (HIV-1) Gag, Gag/Pol and Rev proteins include pCMV-dR8.2 dvpr. Suitable plasmids encoding for the vesicular stomatitis virus (VSV) G envelope protein include pMD2.G.VSV.G.
In a preferred embodiment of the method of preparation of T cells secreting the bispecific antibody of the invention, the method comprises:
In the previous embodiment, lentiviral particles are produced using a 4-plasmid system, alternatively known as a third generation lentiviral vector.
Suitable lentiviral vectors encoding the bispecific antibody of the invention include pCCL. Suitable plasmids encoding the human immunodeficiency virus 1 (HIV-1) Gag and Gag/Pol proteins include pMDLg/RRE. Suitable plasmids encoding the HIV-1 Rev protein include pRSVrev. Suitable plasmids encoding the vesicular stomatitis virus (VSV) G envelope protein include pMD.G.
The plasmid pCCL comprises the sequence encoding the bispecific antibody of the invention and the cytomegalovirus (CMV) promoter.
In an embodiment, cells used in step (i) are human cells. In a preferred embodiment, cells used in step (i) are HEK293T cells.
In an embodiment, step (i) further comprises adding a compound selected from the group consisting of calcium phosphate, cationic polymers, DEAE-dextran, polyethylenimine (PEI), liposomes and polymeric gene carriers (polyplexes).
The method of preparation of T cells of the invention unexpectedly provides T cells comprising 1 or 2 copies per cell genome of the gene encoding the bispecific antibody, which reduces the risk of mutational insertion and increases the safety of a therapy based on said cells.
In an embodiment of the method of preparation of T cells of the invention, the mixture formed in step (i) is incubated for a period from 24 to 48 hours.
In a preferred embodiment of the method of preparation of T cells of the invention, said activating agent is selected from the group consisting of anti-CD3 antibody, anti-CD3-coated beads, anti-CD28 antibody, anti-CD28-coated beads and mixtures thereof.
In a preferred embodiment of the method of preparation of T cells of the invention, the mixture formed in step (iii) is incubated for a period from 24 to 48 hours. In a more preferred embodiment, the mixture formed in step (iii) is incubated for 48 hours.
In a preferred embodiment of the method of preparation of T cells of the invention, the mixture formed in step (iii) is incubated at a T cells:activating agent ratio from 100:1 to 1:3. In more preferred embodiments, said ratio is selected from the values 100:1, 30:1, 10:1, 3:1, 1:1 and 1:3. In a more preferred embodiment, said ratio is 1:3.
In a preferred embodiment of the method of preparation of T cells of the invention, the concentration of the T cells in the mixture formed in step (iii) is from 1×105 to 1×107 cells/ml.
In a more preferred embodiment, the concentration of the T cells in the mixture formed in step (iii) is 1×106 cells/ml.
In a preferred embodiment of the method of preparation of T cells of the invention, the ratio of lentiviral particles:T cells in step (iv) is from 1 to 20, preferably from 1 to 10. In a preferred embodiment of the method of preparation of T cells of the invention, the value of the ratio of lentiviral particles:T cells in step (iv) is selected from 1, 5 and 10.
In a preferred embodiment, the method of preparation of T cells of the invention further comprises the following step:
In a preferred embodiment of the method of preparation of T cells of the invention, said T cells are cultured in step (v) for a period from 6 to 12 days. In a more preferred embodiment of the method of preparation of T cells of the invention, said T cells are cultured in step (v) for a period from 6 to 8 days.
The present invention also provides T cells secreting the bispecific antibody of the invention.
The present invention also provides T cells obtainable according to the method of preparation of T cells of the invention.
The examples evidence that T cells secretes the A3B1-OKT3 bispecific antibody and that this bispecific antibody bound to the CD3 complexes on the cell surface of the T cells. This results in an effective and specific adhesion of T cells to plastic-immobilized CD19.
As shown in the examples, T cells secreting the A3B1-OKT3 bispecific antibody unexpectedly recognize and kill CD19-positive target cells in antigen dependent manner more efficiently, at lower E:T ratios, than CD19 CAR-T cells transduced at the same ratio of lentiviral particles:targets (Multiplicity of Infection, MOI) (Example 3 and
In the examples, T cells secreting the bispecific antibody A3B1-OKT3 (STAb-T cells) were mixed with other T cells (bystander T cells). STAb-T cells activated bystander T cells and hence, activated bystander T cells then recognize and kill CD19+ target cells. This has been observed at a ratio (A-T:Th0) of STAb-T cells:bystander T cells of 1:1000 (Example 4), which resembles tumors, where a reduced number of T cells secreting bispecific antibody could activate the entire population of tumor infiltrating lymphocytes (TIL) to induce an effective anti-tumor response. This feature represents a substantial increase in the therapeutic potential of the T cells of the invention with respect to CAR-T cell therapies, which is a clear advantage.
As shown in the examples, A3B1-OKT3 bispecific antibody secreted by STAb-T cells activated bystander T cells that were separated from STAb-T cells by a semipermeable membrane (a physical barrier for the T cells) (Example 4).
As shown in the examples, it has been observed that CAR-mediated immunological synapse (IS) exhibited a disorganized multifocal signaling cluster structure, with major differences relative to the typical TCR-initiated IS. It has been shown that A3B1-OKT3 bispecific antibody and blinatumomab configure a canonical IS, with actin clearance and CD3 accumulation in the central supramolecular activation dusters (cSMAC) (Examples 5 and 7). These results indicate that IS topology positively influence the functional capacity and cytotoxic potential of CAR-T and STAb-T cells, representing an advantage of STAb-T cells secreting the bispecific antibody of the invention over CAR-T cells in cancer immunotherapy.
As shown in the examples, data from signaling experiments showed shorter activating signals in CAR-Jurkat (CAR-JK) cells compared to control Staphylococcus Aureus Enterotoxin-E (SEE)-stimulated cells (Example 6). This might be related to the shorter time required for a CAR to form a functional IS with respect to the time required by the TCR. Thus, CAR T cells dissociate faster than TCR-stimulated T cells from killed tumor targets, initially enabling faster serial killing. However, far from giving rise to more efficient tumor clearance, CAR-mediated killing is eventually reduced compared to that mediated by TCR ligation, due to a sharp CAR downregulation. On the other hand, as T cell exhaustion occurs after repeated activation of T cells, it may be argued that this faster kinetics of serial killing would render CAR-T cells more rapidly prone to exhaustion. By contrast, the interaction of the T cells secreting the bispecific antibody of the invention with target cells elicited signaling kinetics more similar to those generated by activation controls. Altogether, it can be derived from these results that the T cells secreting the bispecific antibody of the invention give rise to a T cell-early activation reminiscent of the one induced by T cells stimulated by antigen during physiological cognate interactions.
As shown in the examples, in real-time cytotoxicity assays, STAb-T cells killed leukemia cells more efficiently than CD19 CAR-T cells at low effector:target (E:T) ratios (Example 8).
Importantly, it has been shown, in long-term co-culture experiments performed with low E:T ratios, that CAR-T cells were not able to completely abrogate tumor cell growth, and this might be related to the loss of CD19 cell-surface expression, what would render tumor cells “invisible” to CAR-T cells. By contrast, STAb-T cells secreting the bispecific antibody of the invention efficiently killed tumor cells in a short period of time, even at the lowest E:T ratio. It is stated that STAb-T cells are more effective than CAR-T cells in preventing tumor relapse. Moreover, the observed sharp downmodulation of CAR expression after encountering CD19+ cells may help even CD19-expressing targets to escape T cell control. On the contrary, the high levels of both, CD3 expression and A3B1-OKT3 bispecific antibody secretion demonstrated after the STAb-T cell of the invention—tumor cell interaction, explain, together with the ability to recruit bystander T cells, the observed higher anti-tumor effect of CD19 STAb-T of the invention over CD19 CAR-T cells.
The T cells of the invention provide cancer therapies with several advantages. First, in vivo secretion of the bispecific antibody of the invention by the T cells of the invention results in effective concentrations of T cell-redirecting bispecific antibodies, which compensates for the rapid renal clearance of small-sized antibody fragments and render better tolerated bispecific antibodies. Second, in vivo secretion of the bispecific antibody of the invention by the T cells of the invention removes potential concerns about formulating and long-term storage of bispecific antibodies in a manner that prevents aggregation and deterioration. Third, T cell recruitment is not restricted to engineered T cells of the invention, as is the case with the CAR-T cell approach. The recruitment, by bispecific antibodies, of both engineered T cells of the invention and unmodified bystander T cells, present at the tumor site, leads to an improvement in anti-tumor T cell responses. Fourth, STAb-T strategy promotes a canonical IS topology which may be a determining factor in order to increase the functional capacity and cytotoxic potential of the STAb-T cells secreting the bispecific antibody of the invention. Fifth, the interaction between CD19 and CAR promotes an intense and rapid downmodulation of both molecules, which could promote the escape of leukemic cells from immune control. This situation is not observed in co-cultures of CD19+ leukemic cells and the STAb-T cells secreting the bispecific antibody of the invention.
In a preferred embodiment, the T cells of the invention comprise 1 or 2 copies per cell genome of the synthetic polynucleotide encoding the bispecific antibody of the invention or vector comprising the same. Said T cells comprising 1 or 2 copies per cell genome of the gene encoding the bispecific antibody reduce the risk of mutational insertion and increases the safety of a therapy based on said cells.
In an embodiment, the T cells of the invention comprise the synthetic polynucleotide encoding the bispecific antibody of the invention or a vector comprising said synthetic polynucleotide and secretes the specific antibody of the invention.
The present invention also provides the bispecific antibody, or the T cells of the invention, for use as a medicament.
The present invention also provides the bispecific antibody, or T cells of the invention, for use in the treatment of a hematological malignancy in a subject.
In an embodiment of the bispecific antibody, or T cells, for use of the invention, said hematological malignancy is selected from the group consisting of lymphoma, leukemia and myeloma.
In a preferred embodiment of the bispecific antibody, or T cells, for use of the invention, said hematological malignancy is selected from the group consisting of B cell acute lymphoblastic leukemia (ALL), minimal residual disease-positive ALL, chronic lymphatic leukemia (CLL), hairy cell leukemia, non-Hodgkin's lymphoma, B cell lymphoma, mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone B cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, myelodysplastic syndrome (MDS) and multiple myeloma.
In an embodiment, the present invention refers to the use of the bispecific antibody, or the T cells, of the invention for the manufacture of a medicament.
In another embodiment, the present invention refers to the use of the bispecific antibody, or T cells, of the invention for the manufacture of a medicament for the treatment of a hematological malignancy in a subject.
In an embodiment, the present invention also refers to a method of treating a subject with a hematological malignancy which comprises administering the bispecific antibody, or T cells, of the invention to said subject.
In a preferred embodiment, the present invention refers to a method of treating a subject with a hematological malignancy which comprises administering an effective amount of the bispecific antibody, or T cells, of the invention to said subject.
In embodiments, said hematological malignancy is selected from the group consisting of lymphoma, leukemia and myeloma.
In embodiments, said hematological malignancy is selected from the group consisting of B cell acute lymphoblastic leukemia (ALL), minimal residual disease-positive ALL, chronic lymphatic leukemia (CLL), hairy cell leukemia, T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, myelodysplastic syndrome (MDS) and multiple myeloma.
In a preferred embodiment of the bispecific antibody, or T cells, for use of the invention, said bispecific antibody, or T cells, are administered to a subject by infusion or injection.
In a preferred embodiment, combination therapies comprising CAR-T cells and T cells of the invention are provided, to obtain improved therapeutic effects in cancer patients. In a more preferred embodiment, combination therapies comprising CAR-T cells and T cells of the invention provide synergistic therapeutic effects in cancer patients.
Therefore, in a preferred embodiment of the bispecific antibody or T cells for use of the invention, said bispecific antibody, or T cells, are combined with a treatment comprising administering CAR-T cells to said subject.
In a preferred embodiment, T cells of the invention are combined with CAR-T cells with the same or different specificity.
In a more preferred embodiment of the bispecific antibody, or T cells, for use of the invention, said CAR-T cells are anti-CD19 CAR-T cells.
In a yet more preferred embodiment of the bispecific antibody, or T cells, for use of the invention, said anti-CD19 CAR-T cells comprise the anti-CD19 light chain variable domain VLCD19 consisting of the amino acid sequence SEQ ID NO: 3 and the anti-CD19 heavy chain variable domain VHCD19 consisting of the amino acid sequence SEQ ID NO: 5.
In a preferred embodiment of the bispecific antibody, or T cells, for use of the invention, said T cells and said CAR-T cells are administered to said subject sequentially or simultaneously.
The present invention also provides a pharmaceutical composition comprising the bispecific antibody, or T cells, of the invention, and at least one pharmaceutically acceptable excipient.
The pharmaceutical composition of the invention may consist of water solutions, saline solutions or solutions buffered to physiological pH. The pharmaceutical composition may include various carrier agents, thickeners, buffers, preservatives, surfactants, nanoparticles, liposomes and others. The pharmaceutical composition may further include active ingredients such as antimicrobial agents, anti-inflammatory agents, anaesthetic agents, etc.
The pharmaceutical composition of the invention may be administered to a subject in several different ways, depending on whether the treatment is local or systemic, and depending on the area to be treated. Thus, for example, pharmaceutical composition of the invention may be administered to a subject by ocular, vaginal, rectal, intranasal, oral, by inhalation, or by parenteral route, whether intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intra-arterial, intralymphatic, intravenous, intrathecal, intra-ocular, intracranial, intraventricular, intratracheal and intratumoral. Parenteral administration, if used, is generally performed by injection. The solutions for injection can be prepared in various ways, such as solutions or liquid suspensions.
The excipient may be an inert ingredient or ingredients. As an example, said excipient may be a diluent.
The pharmaceutical composition of the invention can be administered with or without combining with other active ingredients.
The pharmaceutical composition of the invention can be administered in single or multiple doses.
The present invention also provides a synthetic polynucleotide encoding the bispecific antibody of the invention, or a vector comprising the same.
In an embodiment of the synthetic polynucleotide or a vector comprising the same, of the invention, said synthetic polynucleotide is at least 95% identical to the polynucleotide sequence SEQ ID NO: 20.
In a preferred embodiment of the synthetic polynucleotide or a vector comprising the same, of the invention, said synthetic polynucleotide of the invention consists of the polynucleotide sequence SEQ ID NO: 20.
Material and Methods
Cell Lines and Culture Conditions
HEK293 (CRL-1573), HEK293T (CRL-3216), and HeLa (CCL-2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Walkersville, Md., USA) supplemented with 2 mM L-glutamine (Life Technologies, Paisley, UK), 10% (vol/vol) heat inactivated fetal calf serum (FCS) and antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) (both from Sigma-Aldrich, St. Louis, Mo., USA), referred as to DMEM complete medium (DCM). Jurkat, Clone E6-1 (TIB-152), Raji (CCL-86), NALM6 (CRL3273) and K562 (CCL-243) cells were maintained in RPMI-1640 (Lonza) supplemented with 2 mM L-glutamine, heat-inactivated 10% FCS, and antibiotics, referred as to RPMI complete medium (RCM). All cell lines were obtained from the American Type Culture Collection (Rockville, Md., USA), and were grown at 37° C. and 5% CO2. NALM6 cells expressing firefly luciferase (Luc) (NALM6Luc) were provided by Dr. Manel Juan (Department of Immunology, Hospital Clinic, Barcelona, Spain). HeLaLuc are HeLa cells infected with lentivirus encoding the Luc gene. All cell lines were routinely screened for mycoplasma contamination by PCR using the Mycoplasma Gel Detection Kit (Biotools, Madrid, Spain).
Jurkat cells were derived from the peripheral blood of a subject with T cell leukemia. Nalm6 cells were derived from the peripheral blood of a subject with acute lymphoblastic leukemia (ALL). Raji cells were derived from the B-lymphocytes of a subject with Burkitt's lymphoma.
Vector Construction and Preparation of Lentiviral Particles
To construct the expression vector pCDNA3.1-A3B1-OKT3 a synthetic gene (A3B1-OKT3 BiTE) encoding the human kappa light chain signal peptide L1, the A3B1 scFv (VL- VH), a five-residue linker (G4S), the OKT3 scFv (VH- VL), and a C-terminal polyHis (HHHHHH) tag was synthesized by GeneArt AG (ThermoFisher Scientific, Regensburg, Germany), and cloned as HindIII/XbaI into the plasmid pCDNA3.1 (ThermoFisher Scientific). To generate the lentiviral transfer vector the complete A3B1-OKT3 bispecific antibody gene (including signal peptide L1, A3B1 scFv, (G4S), OKT3 scFv and polyHis tag) was synthesized by GeneArt AG and cloned as MluI/BspEI into the vector pCCL-EF1α-CAR19, to obtain the plasmid pCCL-EF1α-A3B1 BiTE. To produce lentiviral particles, HEK293T cells were transfected with the transfer vector together with packaging plasmids pMDLg/pRRE (Addgene, 12251), pRSVrev (Addgene, 12253), and envelope plasmid pMD2.G (Addgene, 12259), using linear polyethyleneimine (PEI) molecular weight (MW) 25,000 (Polysciences, 23966-1).
HEK293T cells (6×106) were plated 24 hours before transfection in 10-cm dishes. At the time of transfection, 14 μg total DNA (6.9 μg transfer vector, 3.41 μg pMDLg/pRRE, 1.7 μg pRSV-Rev, and 2 μg pMD2.G) was diluted in serum-free DMEM. 35 μg PEI was added to the mix and incubated for 20 min at room temperature. After incubation, DNA-PEI complexes were added onto the cells cultured in 7 mL complete DMEM. Media were replaced 4 hours later. Viral supernatants were collected 48 hours later and clarified by centrifugation and filtration using a 0.45-μm filter. Viral supernatants were concentrated using ultracentrifugation at 26,000 rpm for 2 hours 30 min. Virus-containing pellets were resuspended in complete XVivo15 media and stored at −80° C. until use.
Viral supernatants were collected 48 hours later and clarified by centrifugation and filtration using a 0.45 μm-pore filter, ultracentrifuged (26,000 rpm for 2 hours), resuspended in complete XVivo15 media (Lonza, Walkersville, Md., USA), aliquoted and stored at −80° C. until use.
Expression and Purification of the A3B1-OKT3 Bispecific Antibody
HEK293 cells were transfected with the expression vector using calcium phosphate and selected in DCM with 500 μg/ml G-418 (Life Technologies, Grand Island, N.Y., USA) to generate stable cell lines. Supernatants from transiently and stably transfected cell populations were analyzed by western blotting and FACS. The A3B1-OKT3 bispecific antibody was purified from DCM conditioned medium collected from stably transfected HEK293 cells. Collected medium was centrifuged, filtered and purified 500 mL at a time using a 1 mL HiTrap Excel column (GE Healthcare, Uppsala, Sweden), washing with 30 column volume of 20 mM imidazole in PBS (pH 7.4) and eluting with 500 mM of imidazole in PBS. Elution fractions containing the relevant proteins were then pooled, dialyzed against PBS, and concentrated using spintrap columns (Merck Millipore, Billerica, Mass., USA).
Western Blotting
Samples were separated under reducing conditions on 10-20% Tris-glycine gels (Life Technologies, Carlsbad, Calif., USA) and transferred onto PVDF membranes (Merck Millipore, Tullagreen, Carrigtwohill, Ireland) and probed with anti-His mAb (Quiagen, Hilden Germany) (200 ng/ml), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse (GAM) IgG, Fc specific (1:5000 dilution) (Sigma-Aldrich). Visualization of protein bands was performed with Pierce ECL Western Blotting substrate (Rockford, Ill., USA).
For analysis of BiTE and CAR expression, samples were lysed for 5 minutes in ice-cold RIPA buffer (Sigma-Aldrich) with 5 mM EDTA and 1× Halt Protease Inhibitor Cocktail (ThermoFisher). Lysates were then centrifuged at 11,000 g for 10 minutes at 4° C. and soluble fractions were collected. For analysis of BiTE secretion by transfected or transduced cells, supernatants were collected. 15 mg of protein or 16 ml of supernatant were separated under reducing conditions on 10-20% Tris-glycine gels (Life Technologies, Carlsbad, Calif., USA), transferred onto lmmobilon-PVDF membranes (Merck Millipore, Tullagreen, Carrigtwohill, Ireland) and probed with mouse anti-human CD247 (1:1000) (BD Biosciences) or anti-His mAb (Qiagen, Hilden Germany) (200 ng/ml), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse (GAM) IgG, Fc specific (1:5000) (Sigma-Aldrich). Mouse anti-b actin HRP-conjugated mAb (1:50,000) was used as loading control. Visualization of protein bands was performed with Pierce ECL Western Blotting substrate (Rockford, Ill., USA).
For analysis of T cell signaling, CAR-JK and STAb-JK cells were incubated at 37° C. with Raji cells at a Jurkat/Raji ratio 10:1 for the indicated times. NT-JK cells were incubated with non-loaded Raji cells or Raji cells loaded during 1 hour with 5 nM Blincyto (blinatumomab) (Amgen Inc, Thousand Oaks, Calif.) or 1 μg/mL Staphylococcus Aureus Enterotoxin-E (SEE) (Toxin Technologies, Sarasota, Fla., USA). The stimulation time 0 minutes corresponds to Jurkat cells mixed with Raji cells at room temperature and immediately centrifuged and lysed. Samples were lysed for 30 minutes in ice-cold lysis buffer containing 20 mM Tris-HCl pH 7.5; 1% NP-40; 0.2% Triton X-100 (Sigma-Aldrich); 2 mM EDTA; 150 mM NaCl; 1.5 mM MgCl2; 5 mM β-glicerolphosphate; lx protease inhibitor cocktail; 1 mM NaF; 1 mM PMSF; 1 mM Na3VO4 and 1 mM sodium pyrophosphate. Lysates were then centrifuged at 10.000 rpm for 10 minutes at 4° C. and soluble fractions were collected, mixed with 6× Laemmli buffer (Alfa Aesar, Haverhill, Mass., USA) containing 20% β-mercaptoethanol, boiled at 95° C. for 5 minutes and resolved in 10% SDS-PAGE acrylamide gels. Resolved proteins were transferred to Immobilion PVDF membranes, which were blocked with blocking buffer (LI-COR Bioscience, Lincoln, Nebr., USA), incubated over night with rabbit anti phospho-Y783-PLCγ1, anti PLCγ1, anti phospho-T202/T204-ERK1/2 or mouse anti-ERK1/2 primary antibodies (all from Cell Signaling Technology, Beverly, Mass., USA) and incubated 30 minutes with 680 goat-anti rabbit and 800 goat-anti mouse IR dyes (Miltenyi Biotec). All blots were scanned, and fluorescence was quantified with an Odyssey Infrared Imager (LI-COR). Densitometry of images was done with Image Studio Freeware (LI-COR). When necessary, blots were striped in 50 ml containing 2% SDS; 12.5% Tris-HCl pH 6.8 and 0.7% μ-mercaptoethanol for 30 minutes at 50° C.
Enzyme-Linked Immunosorbent Assay
Human CD19:human Fc chimera (CD19:Fc) (R&D Systems, Minneapolis, Minn., USA) was immobilized (5 μg/ml) on Maxisorp plates (NUNC, Roskilde, Denmark) overnight at 4° C. After washing and blocking, conditioned media or purified protein solution were added and incubated for 1 hour at room temperature. The wells were washed and anti-His mAb (Qiagen) added (1 μg/ml). After washing, HRP-GAM IgG, Fc specific (1:2000 dilution) (Sigma-Aldrich) was added, and the plate was developed using tetramethylbenzidine (TMB) (Sigma-Aldrich).
Flow Cytometry
The following mAbs against human proteins, were used: FITC-conjugated anti-CD3 (clone UCHT1), APC-conjugated anti-CD3 (clone UCHT1), PE/Cy7-conjugated anti-CD8 (clone RPA-T8), PE-conjugated anti-CD69 (clone L78), all from BD Biosciences (San Jose, Calif., USA); PC7-conjugated anti-CD19 (clone J4.119, Beckman Coulter, Marseille Cedex, France); and PE-conjugated anti-CD4 (clone MEM-241, Immunotools, Friesoythe, Germany). Dapi (Sigma-Aldrich) was used as viability marker. Cell surface expression of A3B1 CAR was analyzed using an APC-conjugated F(ab′)2 goat anti-mouse IgG F(ab′)2 (Jackson ImmunoResearch, West Grove, Pa., USA). Cell surface-bound A3B1-OKT3 bispecific antibody was detected with 1 μg/ml anti-6×His tag-biotin mAb (clone HIS.H8, Invitrogen, Rockford, Ill., USA), and PE-conjugated streptavidin (BD Biosciences). Cell acquisition was performed in a BD FACSCAnto II flow cytometer using BD FACSDiva software (both from BD Biosciences, San Jose, Calif., USA). Analysis was performed using FlowJo V10 software (Tree Star, Ashland, Oreg., USA).
Lentivirus Titration
All lentivirus stocks were normalized for p24 and RNA. The p24 concentration was determined by ELISA (Takara, Saint-Germain-en-Laye, France), and the genomic lentiviral RNA by qRT-PCR (Takara). In the case of A3B1 CAR-encoding lentivirus, functional titers (TU/ml) were determined by FACS analysis after limiting dilution in HEK293T cells, using an APC-conjugated F(ab′)2 fragment goat anti-mouse IgG F(ab′)2 fragment specific (Jackson ImmunoResearch Laboratories).
T Cell Transduction and Culture Conditions
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of volunteer healthy donors by density gradient centrifugation using Lymphoprep solution (Axis-Shield, Oslo, Norway). All donors provided written informed consent in accordance with the Declaration of Helsinki. CD3+ T cells were purified by negative selection using the Pan T Cell Isolation Kit, human and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. The purity of isolated populations was routinely >95%. Cells were then activated and expanded for 24 hours using anti-CD3/CD28 beads (Dynabeads, Gibco) at 1:3 cell:bead ratio in RCM, at a concentration of 1×106 cells/ml. 24 hours later cells were left untransduced (NT cells) or transduced with A3B1 CAR (CAR-T cells) or A3B1-OKT3 bispecific antibody (STAb-T cells) encoding lentivirus at the indicated MOI (Multiplicity of Infection, ratio of lentiviral particles:targets). A3B1 CAR-T cells were transduced with a lentiviral vector encoding an anti-CD19 second generation 4-1BB/CD3 CAR (Castella et al, 2019). A period of cell expansion of 6-8 days was carried out before conducting experiments. Three different cell transductions using three different PBMC donors were used to conduct the experiments in triplicate.
T Cell Activation Assays
For Jurkat T cell activation assays, 105 Jurkat cells were co-cultured with Raji, Nalm6 or K562 cells at effector:target (E:T) ratio of 2:1, and 50 μl of supernatant from transfected HEK293 cell cultures was added. After 48 hours, CD69 expression was analyzed by flow cytometry, and culture supernatants were collected and analyzed for IL-2 levels by ELISA (Diaclone, Besançon Cedex, France.) For primary T cell activation assays, anti-CD3/CD28 beads were removed five days after lentiviral transduction, and activated T cells (A-T) were left resting for 24 hours at a concentration of 0.8×106 cells/ml. Then, transduced or non-transduced A-T were co-cultured with freshly isolated T cells (non-activated T cells, Th0) from the same donor, and Nalm6 or HeLa target cells at the indicated ratios. As controls, Th0 cells were cultured with Nalm6 or Hela target cells in the absence or the presence of 100 ng/ml blinatumomab. After 48 hours, supernatants were collected and levels of IFN-γ were analyzed by ELISA (Diaclone). For proliferation assays, de-beaded A-T were stained with 2.5 μM Cell Trace Violet, and freshly isolated T cells were stained with 2.5 μM Cell Trace CFSE (both from Life Technologies, Eugene, Oreg., USA) following manufacturer's instructions. Stained A-T and Th0 cells were co-cultured with Nalm6 or HeLa target cells at the indicated ratios. After 5 days, samples were acquired in a FACSCanto flow cytometer and T cell proliferation was analyzed using FCS Express 6 Plus Software (De Novo Software, Los Angeles, Calif., USA).
Cytotoxicity Assays
As indicated above, five days after lentiviral transduction, anti-CD3/CD28 beads were removed and A-T cells were left resting for 24 hours. Then, transduced or non-transduced A-T cells were co-cultured with freshly isolated Th0 cells and NALM6Luc or HeLaLuc target cells at the indicated ratios. As controls, Th0 cells were cultured with Nalm6 or Hela target cells in the absence or the presence of 100 ng/ml blinatumomab. After 48 hours viability was measured adding D-luciferin (Promega, Madison, Wis., USA) to a final concentration of 20 μg/ml and bioluminescence quantified in relative light units using a Victor luminometer (PerkinElmer, Waltham, Mass., USA). A 100% lysis control was included by treating the target cells with 1% Triton-X100 (Sigma-Aldrich), and the value for spontaneous lysis was obtained by incubating the target cells with effector cells (NA-T) only. Percent tumor cell viability was calculated as the mean bioluminescence of each sample divided by the mean bioluminescence of the input number of control target cells times 100. Specific lysis is the difference in tumor cell viability relative to control (0%).
For cytotoxic studies in transwell assays, polycarbonate filter inserts (4.26 mm diameter) with 0.4 μm pores (Corning, Kennebunk, Me., USA) were used. Nalm6Luc and HeLaLuc/MKN45Luc cells (5×104) were plated on bottom wells of 96-well plate with 2:1 E:T ratio (105 de-beaded A-T cells: NT, CAR-T or STAb-T cells at the indicated ratios were added to insert wells). After 48 hours bioluminescence quantified as described above and cell culture supernatants were collected for IFN-γ secretion analysis.
For realtime cytotoxicity assays, wild type HEK293 (HEK WT) or HEK293 cells stably-transfected to express membrane-bound CD19 (HEK-CD19) were plated at a density of 104 cells per well in a volume of 50 μL in an E-Plate 16 (Acea Biosciences, San Diego, Calif.). After plating, cells were cultured for 20 h in an xCELLigence RTCA DP system (Acea BioSciences), inside a cell culture incubator at 37° C. and 5% CO2, with measurements taken at 15 min intervals to measure Cell Index, a measure of electrical impedance across well electrodes resulting from cell adherence. After 20 h, NT-T, STAbT or CAR-T cells, at different E:T ratios, were added in a volume of 100 μL per well, with 2 replicate wells per condition. Plates were returned to the xCELLigence and the cytotoxicity was evaluated over 72 h with measurements taken at 15 min intervals. Cell Index values were determined and normalized using RTCA Software 2.0 (Acea Biosciences). Percentage specific lysis was calculated using the following equation: Percentage=[(cell index of UTD−cell index of CAR-T cells)/(cell index of UTD)]×100.
Immunofluorescence and Confocal Microscopy
NT, CAR and STAb, Jurkat or primary, T cells were incubated for 15 minutes on Poly-L-lysine (Sigma-Adrich)-coated coverslips at 37° C. with Raji or Nalm6 target cells at a T cell/target ratio 1:1. Where indicated, target cells were loaded for 1 hour with 5 nM blinatumomab or 1 μg/mL SEE as in Western blot experiments. In order to properly find cell conjugates, target cells were pre-incubated with the fluorescent tracker chloromethyl derivative of aminocoumarin (CMAC) 1 μM (Molecular Probes, Eugene, Oreg., USA). Jurkat/Raji cell conjugates (200.000 cells each) were fixed with 4% paraformaldehyde in PBS for 5 minutes at RT, permeabilized with 0.1% Triton X-100 for 5 minutes at RT and blocked with 10 μg/ml human gamma globulin for 20 minutes at RT. Samples were stained with mouse anti-CD3ε (T3b clon; kindly provided by Dr. Francisco Sanchez-Madrid, (Hospital Universitario de la Princesa, Madrid, Spain) or with Phalloidin-647 (Molecular Probes) (for filamentous actin detection) for 1 hour at RT. Cells were then washed with TBS and incubated with goat anti-mouse-Ig Alexa 488 (Molecular Probes) secondary antibody at RT for 30 minutes. Finally, coverslips were washed with TBS and distilled water before being mounted with Mowiol (Sigma-Aldrich) medium.
Confocal sections of fixed samples were acquired using an SP-8 laser scanning laser confocal microscopy (Leica Microsystems, Wetzlar, Germany), with a 60×/1.35 oil immersion objective. CMAC, A488 and phalloidin-647 were excited by 405, 488 and 633 nm laser lines, respectively. For 3D reconstructions, z-stacks through the complete IS were acquired every 0.3 μm. Actin clearance was estimated by the ratio of the area of the central region of the IS depleted of actin versus the complete area of the IS including the actin ring in 3D images. Assessment of CD3 coalescence and central supramolecular activation dusters (cSMAC) formation was assessed by visual inspection of the 3D images. 3D reconstruction and image quantitation were performed with ImageJ freeware (National Institutes of Health, Rockville, Md., USA).
Statistical Analysis
Results of experiments are expressed as mean±standard deviation (SD). Statistical tests were done with Prism 6 (GraphPad Software, USA).
An anti-CD19/anti-CD3 BiTE antibody, identified herein as A3B1-OKT3 bispecific antibody or as A3B1-OKT3 BiTE, was generated. A3B1-OKT3 bispecific antibody comprises the anti-CD19 A3B1 scFv linked to the anti-CD3 OKT3 scFv in a tandem structure (
The anti-CD19 A3B1 scFv and the anti-CD3 scFv are separated by a linker (linker 2). The anti-CD19 A3B1 scFv comprises an anti-CD19 light chain variable domain VLCD19 and an anti-CD19 heavy chain variable domain VHCD19. The anti-CD3 scFv comprises an anti-CD3 heavy chain variable domain VHCD3 and an anti-CD3 light chain variable domain VHCD3. The domains are arranged in the order VLCD19-VHCD19-VHCD3-VLCD3.
The domains VLCD19-VHCD19 are separated by a linker (linker 1) and the domains VHCD3-VLCD3 are also separated by a linker (linker 3).
A3B1-OKT3 bispecific antibody further comprises the human kappa light chain signal peptide L1 and a polyhistidine tag. Table 1 provides details of A3B1-OKT3 bispecific antibody sequence features. The 3 CDRs regions of each light and heavy variable domains are identified in Table 1.
The A3B1-OKT3 bispecific antibody was secreted by transfected HEK293 cells, and the migration pattern was consistent with the molecular weight calculated from its amino acid sequence (54.2 kDa without the signal sequence;
FACS analysis demonstrated that A3B1-OKT3 BiTE bound to CD3+ Jurkat cells and to CD19+ Nalm6 and Raji cells, but not to CD3-CD19- K562 cells (
The A3B1-OKT3 bispecific antibody was purified from conditioned medium from stably transfected HEK293 cells by immobilized metal affinity chromatography, which yields protein (1 mg/L) that was >90% pure, as assessed by SDS/PAGE (
The A3B1-OKT3 bispecific antibody was cloned into a lentiviral vector and Jurkat T cells transduced at MOI (Multiplicity of Infection, ratio of lentiviral particles:targets) values of 1, 5 and 10, with A3B1-OKT3 bispecific antibody-encoding or A3B1 CAR-encoding lentiviruses and the relationship between the number of vector integrations and transgene expression analyzed. Vector copy number (VCN) was found to be similar in both cases, between 1 and 5 copies for A3B1 CAR-infected Jurkat T cells (CD19 CAR-Jurkat), and 1 and 7 for A3B1-OKT3 bispecific antibody-infected Jurkat T cells (CD19 STAb-Jurkat) (
Next, CD3/CD28-activated primary human T cells were transduced to generate CD19 STAb-T cells. Activated T cells were transduced at a MOI (Multiplicity of Infection, ratio of lentiviral particles:targets) of 10 with A3B1-OKT3 bispecific antibody-encoding or A3B1 CAR-encoding lentiviruses, and after an expansion period, the expression of both A3B1-based molecules analyzed. The A3B1-OKT3 bispecific antibody was detected in conditioned-medium from STAb-T cells, had the expected molecular weight (
T cells secreting A3B1-OKT3 bispecific antibody (STAb-T cells) secreted IFNγ after a co-culture at a 2:1 E:T ratio with CD19+ Nalm6 cells, but not with CD19- HeLa cells (
To demonstrate that STAb-T cells were able to recruit bystander T cells to kill CD19-positive targets, different in vitro co-culture setups, with and without direct cell-cell contacts were used (
When stimulated with Nalm6 cells, in a direct cell-cell contact context (
The percentage of dividing T cells was higher than observed when Th0 cells were cultured with Nalm6 cells in the presence of blinatumomab. This ability to recruit bystander Th0 cells results in STAb-T cells being able to induce 100% of tumor cell lysis, even when they represented 1 out of 500 total Target cells (
To further demonstrate that STAb-T cells are able to recruit bystander Th0 cells to CD19-positive targets, transwell assays were used (
The spatial topology of the immunological synapse (IS) assembled by the A3B1 CAR or the A3B1-OKT3 bispecific antibody was studied in CD19 CAR-Jurkat (CD19 CAR-JK) cells and CD19 STAb-Jurkat (CD19 STAb-JK) cells, using the CD19-expressing cell line Raji as antigen presenting cell. As controls, non-transduced Jurkat (NT-JK) cells were incubated with unloaded (non-activated control) or with Raji cells loaded with blinatumomab or the bacterial superantigen (SAG) staphylococcal enterotoxin E (SEE) (activation controls). Bacterial SAGs bind, as intact molecules to the class II MHC molecules expressed on professional antigen presenting cells (APCs) outside the peptide-binding groove then sequentially bind the TCR via the variable region of the TCR β-chain. Jurkat—Raji cell conjugates incubated at 37° C. for 15 minutes were stained for F-actin and CD3ε to evaluate the organization of the distal and central supramolecular activation clusters, dSMAC and cSMAC, respectively. 3D confocal microscopy was implemented to visualize the central filamentous actin clearance, with the typical actin ring at the dSMAC, and the coalescence of CD3ε microclusters at the cSMAC occurring in the mature IS. All conditions, including CD19 CAR-JK cells, recruited CD3ε to the IS. However, STAb-JK, but not CAR-JK cells cleared actin and formed the cSMAC by CD3 coalescence in a similar way to J-NT-T cells stimulated by SEE or blinatumomab (
The activation of CAR-JK and STAb-JK cells was assessed in co-cultures with Raji cells. As activation control, NT-T cells were stimulated with blinatumomab- or SEE-loaded Raji cells. As negative control, NT-JK cells were incubated with Raji cells alone. PLCγ1 and ERK1/2 activation were analyzed by Western blot due to their important role in early activation signaling downstream the TCR. Interestingly, STAb-JK cells showed PLCγ1 and ERK1/2 activation kinetics similar to NT-JK cells stimulated with SEE or blinatumomab. However, CAR-JK cells showed a more transient signaling compared to the rest of stimulation conditions (
The immunological synapse assembly by primary STAb-T cells and CAR-T cells following their interaction with Raji cells was analyzed. Primary STAb-T cells organized a canonical IS, with proper filamentous actin (F-actin) containing dSMAC and accumulation of CD3ε at the cSMAC (
In an impedance-based cytotoxicity assay for real-time and label-free assessment of T cell-mediated killing primary STAb-T cells were found to mediate rapid reduction in target cell viability against adherent CD19+ cells (
Next, the ability of CD19+ leukemic B cells to escape from immune control was studied by co-culturing primary CAR and STAb T cells with Nalm6 cells at low E:T ratios. Whereas co-culture with NT-T cells did not inhibit the growth of Nalm6 cells (
To more carefully study leukemia escape, Nalm6 cells were co-cultured at 1:1 E:T ratio with lentivirally-transduced Jurkat T cells, CAR-JK or STAb-JK (
Sequence Listing Free Text
The sequence listing contains free text, which is shown in Table 2.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20382113.7 | Feb 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/053745 | 2/16/2021 | WO |
